Nucleotide vector vaccine for immunization against hepatitis

ABSTRACT

Nucleotide vector comprising at least one gene or one complementary DNA coding for at least a portion of a virus, and a promoter providing for the expression of such gene in muscle cells. The gene may be the S gene of the hepatitis B virus. 
     A vaccine preparation containing said bare DNA is injected into the host previously treated with a substance capable of inducing a coagulating necrosis of the muscle fibres.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/146,072, filed Sep. 2, 1998, now U.S. Pat. No. 6,635,624, which is acontinuation of U.S. application Ser. No. 08/633,821, filed Aug. 2,1996, now abandoned, which claims priority to which claims priority toPCT/FR94/00483, filed Apr. 27, 1994 (French Patent Application No.93/12659, filed Oct, 22, 1993), each of which are incorporated herein byreference in their entirety.

The present application relates to a vector for immunization againsthepatitis.

It is also related to a composition containing this vector.

Immunization by injection of bare DNA into muscle tissues has been theobject of several studies since the beginning of the 1990s.

Thus, ULMER et al. (Science, 259, 1745-1749, 1993) obtained protectionagainst the Influenza virus by induction of the cytotoxic T lymphocytesthrough injection of a plasmid coding for the Influenza A nucleoproteininto the quadriceps of mice. The plasmid used carries either the Roussarcoma virus promoter or the cytomegalo virus promoter.

RAZ et al. (Proc. Natl. Acad. Sci. USA, 90, 4523-4527, 1993) injectedvectors comprising the Rous sarcoma virus promoter and a gene coding forinterleukin-2, interleukin-4 or the β1-type transforming growth factor(TGF-β1). The humoral and cell immune responses of the mice to whichthese plasmids have been intramuscularly administered are improved.

WANG et al. (Proc. Natl. Acad. Sci. USA, 90, 4156-4160, 1993) injected aplasmid carrying a gene coding for the envelope protein of the HIV-1virus into mice muscles. The plasmid injection was preceded by treatmentwith bupivacaine in the same area of the muscle. The authors demonstratethe presence of antibodies capable of neutralizing the HIV-1 virusinfection. However, it will be noted that DNA was injected twice a weekfor a total of four injections.

DAVIS et al. (Compte-Rendu du 28ème Congrès Européen sur le muscle,Bielefeld, Germany, 21-25 September 1992) injected plasmids carrying aluciferase or β-galactosidase gene coding by pretreating the muscleswith sucrose or a cardiotoxin. The authors observed the expression ofluciferase or β-galactosidase.

More recently, an article published in Science et Avenir (September1993, pages 22-25) indicates that WHALEN et DAVIS succeeded inimmunizing mice against the hepatitis B virus by injecting pure DNA fromthe virus into their muscles. An initial injection of snake venom toxin,followed 5 to 10 days later by a DNA injection, is generally cited. Itis specified that this is not a practical method.

These studies were preceded by other experiments in which various DNAswere injected, in particular into muscle tissues. Thus, the PCT/USapplication No. 90/01 515 (published under N^(o) WO-90/11 092) disclosesvarious plasmid constructions which can be injected in particular intomuscle tissues for the treatment of muscular dystrophy. However, thisdocument specifies that DNA is preferentially injected in liposomes.

This also applies to Canadian patent CA-362.966 (published under N^(o)1.169.793) which discloses the intramuscular injection of liposomescontaining DNA coding in particular for HBs and HBc antigens. Theresults described in this patent mention the HBs antigen expression. Thepresence of anti-HBs antibodies was not investigated.

International application PCT/FR 92/00 898 (published under N^(o)WO-93/06 223) discloses viral vectors which can be conveyed to targetcells by blood. These vectors are thus recognized by the cell receptors,such as the muscle cells, and can be used in the treatment of musculardystrophy or of thrombosis.

This application does not relate to immunization against viruses suchas, for example, that of hepatitis B.

Thus, it arises from the state of the art cited that althoughimmunization techniques against hepatitis by injection of bare DNA arealready known, these techniques had many disadvantages which made theirimplementation impractical.

Furthermore, the bare DNA used to vaccinate the mice was pure DNA fromthe virus. This type of treatment can not be considered for humanvaccination due to the risks involved for the patients.

Finally, the earliest experiments in which the injected DNA is containedin liposomes did not demonstrate any immune response.

The applicant has therefore aimed at discovering new vectorconstructions allowing immunization against hepatitis without having adetrimental effect on human health.

He has further aimed at finding an additive for compositions containingthe constructions which would allow an effective degeneration of muscletissue before the DNA injection, and compatible with the requirements ofhuman health.

The applicant has surprisingly shown that it is possible to achieve aneffective and durable level of antibodies much greater than the levelpermitting to obtain in man an efficient and durable immune protectionagainst infection by the hepatitis virus, by administering byintramuscular injection a vector with defined construction, and asubstance capable of inducing a coagulating necrosis of the musclefibres.

The present application thus relates to a nucleotide vector comprisingat least:

-   -   a gene or a complementary DNA coding for at least a part of the        virus protein, and    -   a promoter allowing the expression of this gene in muscle cells.

Said vector may not replicate in these cells.

It may also be replicative, allowing to obtain a high number of copiesper cell and to enhance the immune response.

The vector is also chosen in order to avoid its integration into thecell's DNA, such integrations being known to activate the oncogens andinduce cell canceration.

The vector according to the present invention is advantageously aplasmid of partly bacterial origin and notably carrying a bacterialreplication origin and a gene allowing its selection, such as a gene forresistance to an antibiotic.

This vector may also be provided with a replication origin allowing itto replicate in the muscle cells of its host, such as the replicationorigin of the bovine papilloma virus.

The gene or the complementary DNA included in this vector advantageouslycodes for a structure protein of a virus but it can also code for aregulatory protein.

The gene or complementary DNA carried by this vector can code for aleast a portion of a hepatitis virus protein, in particular hepatitis B,and preferentially the protein HBs, in one of its forms S, S-preS2 orS-preS2-preS1, in which case the gene is gene S.

The virus may also be responsible for another hepatitis such as ahepatitis A or of a non-A, non-B hepatitis, such as a hepatitis C, E ordelta.

The gene or protein sequences for these hepatitis viruses are describedor may be deduced from the following documents:

patent FR-79 21 811, patent FR 80.09.039,

patent EP-81.400.634, patent FR 84.03.564,

patent EP 91.830.479 and the article by Najarian et al. (Proc. Natl.Acad. Sci. USA, 1985, 82, 2627-2631).

The vector may also include genes coding for at least a portion of thegp160 protein of HIV-1 virus associated with the p25 protein, and/or thep55 protein, and/or the p18 protein or at least a gene coding for theRev protein of HIV-1 virus.

The vector may also include instead of a virus protein, a protein from apathogenic micro-organism such as a protein from the bacterium causingdiphtheria, whooping cough, listeriosis, the tetanus toxin etc.

The promoter carried by this vector is advantageously the promoter forthe cytomegalovirus (CMV). It may however be any other promoter whichallows the efficient expression of the gene in the muscle cells.

It may thus be:

-   -   an internal or endogenic promoter, that is a promoter of the        virus from which the gene is taken; such a promoter may be        completed by a regulatory element of the muscle or another        tissue, in particular an activating element,    -   a promoter from a gene of a cytoskeleton protein, in particular        desmine as described by BOLMONT et al. (Journal of        submicroscopic cytology and pathology, 1990, 22, 117-122) et        ZHENLIN et al. (Gene, 1989, 78, 243-254).    -   the promoter from the virus HBV surface genes.

Generally, the promoter may be heterologous to the host, that is notnaturally found in the host, but it is advantageously homologous, whilebeing originally active in a tissue other than the muscle tissue.

In addition to the promoter, the vector may include a terminaltranscription sequence, situated downstream of the gene.

Such vector may be the pCMV/HBS or pRCCMV-HBS plasmid, having the SEQ IDN^(o) 1 sequence, filed under N^(o) I-1370 with the Collection Nationaledes Cultures des Micro-organismes de l'Institut Pasteur (CNCM) on 21Oct. 1993.

It may also be the pRSV/HBS plasmid filed under N^(o) I-1371 with theCNCM on 21 Oct. 1993.

This plasmid has a similar structure to pCMV/HBS but includes the Roussarcoma virus (RSV) promoter instead of the cytomegalovirus (CMV)promoter.

Other plasmids may be:

-   -   pCMVHB-S1.S2.S constructed by inserting the fragment Bgl II-Bgl        II of the S gene, obtained from pCP10, into a pBlueScript vector        modified to contain supplementary cloning sites in the        “polylinker” portion. The fragment containing the S gene was        then removed by KpnI-BssH II digestion then cloned into the        corresponding sites of pcDNA 3 (In vitrogen, Rad Systems Europe        Ltd, Abingdon UK) so as to obtain pCMVHB-S1.S2.S. This plasmid        was filed under N^(o) I-1411 with the CNCM.    -   pCMVHB-S2.S obtained by eliminating the pre-S1 part of the HBS        gene from pCMVHB-S1.S2.S by KpnI/MstI digestion, then by bonding        the two extremities after treatment with S1 nuclease.

pCMVHB-S2.S was filed with the CNCM under No. I-1410.

-   -   pHBV-S1.S2.S, filed with the CNCM under N^(o) I-1409, was        obtained by inserting the S gene Bgl II-Bgl II fragment,        obtained from pCP10, into a pBlueScript vector modified to        contain supplementary cloning sites in the “polylinker” portion.    -   pBS-SKT-S1.S2.S codes for the three envelope proteins S, S-preS₁        and S-preS₁-preS₂ of the HBV virus.

The present invention further relates to nucleotide sequences comprisinga promoter homologous to the host and another regulatory sequence forthe expression of a gene or complementary DNA coding for one of theabove mentioned proteins.

The present invention further relates to a vaccine or medicinecontaining at least one vector, or a nucleotide sequence, such asdefined above.

It further relates to a composition capable of inducing a cytotoxicresponse comprised of at least one nucleotide sequence expressed in themuscle cells and including a promoter such as defined above.

It further relates to a non-lipid pharmaceutical composition forimmunization against a viral infection such as a hepatitis including, onthe one hand, at least a substance capable of inducing a coagulatingnecrosis of the muscle fibres and, on the other hand, a vector such asdescribed above or including one of the nucleotide sequences, completeor partial, such as described above. By partial sequence is meant asequence coding for at least 6 amino acids.

Said substance is advantageously bupivacaine.

Advantageously, said composition is characterized in that the vector isadministered in the muscle of the individual to immunized, at least 5days after the administration of the bupivacaine, and substantially inthe same location.

Such prior administration of bupivacaine surprisingly allows to increasethe effectiveness of the vector administration as well as theimmunization of the individual.

Advantageously, the vector is administered ten days after administrationof bupivacaine, and substantially in the same location of theindividual's muscle.

The present composition may also contain additives which are compatibleand pharmaceutically acceptable.

Such composition is preferentially administered by intramuscularinjection. The injection can be carried out using a syringe designed forsuch use or using a liquid jet gun such as described by FURTH et al.(1992, Anal. Biochem. 205, 365-368).

The quantity of bupivacaine required to obtain sufficient degenerationof the muscle tissue, in order to achieve optimal immunization, is inthe order of 0.10 mg to 10 mg per dose of injected composition.

The quantity of vector to be injected in order to achieve optimalimmunization of the individual against a hepatitis varies according tothe protein coded by the gene carried by the vector. As an indication,between 0.1 and 1000 μg of vectors are injected per individual.

The vectors may be obtained by methods known to those skilled in theart, in particular by synthesis or by genetic engineering methods.

Such methods are those described in particular in the technical manual:

Maniatis T. et al. 198213 Molecular Cloning, A Laboratory Manual, ColdSpring Harbour—Ed. New York.

The present invention is illustrated by, without in any way beinglimited to, the following examples, in which:

FIG. 1 is a schematic representation of pRC/CMV-HBs plasmid.

FIGS. 2A to 2D are schematic representations of pCMVHB-S, pCMVHB-S2.S.,pCMVHB-S1.S2.S and pHBV-S1.S2.S plasmids, respectively.

FIGS. 3, 4 and 5 are schematic restriction maps for pCMVHB-S2.S,pCMVHB-S1.S2.S and pRSV-HBS plasmids, respectively.

FIG. 6 illustrates the secretion of antigenic HBs particles (HBs Ag) inng/ml (ordinates) as a function of the number of days (abscissa) forcells carrying the pCMVHB-S, pCMVHB-S1.S2.S, pHBV-S1.S2.S, pSVS orpCMVHB-S2.S plasmids.

FIGS. 7A and 7B illustrate the determination on some particles in FIG. 6of the presence of the preS₁ and PreS₂ antigens using respectivelyanti-preS₁ and anti-preS₂ antibodies. The formation of antibody-antigencomplexes is shown by the optical density (ordinates), as a function ofantigen concentration.

FIGS. 8A to 8D represent the anti-HBS responses (HBS Ab as ordinate,expressed as mUI/ml) and anti-preS2 (preS2 Ab as ordinate, expressed inO.D.) of mice vaccinated by pCMVHB-S (8A), pCMVHB-S2.S (8B),pCMVHB-S1.S2.S (8C) and pHBV-S1.S2.S (8D), respectively.

FIG. 9 illustrates the antibody response, IgG and IgM immunoglobulins(titre as ordinates), of a mouse vaccinated by pCMVHB-S2.S as a functionof the number of weeks (abscissa).

FIGS. 10A to 10C represent the anti-group and anti-subtype ay responsesinduced by DNA from pCMV-S (DNA) or from the HBS antigen (prot),respectively in mice B10 (10A), BIOS (10B) and B10M (10C).

FIGS. 10D to 10F represent the antigroup responses induced by DNA frompCMV-S (DNA) or from the HBS antigens (prot), respectively in mice B10(10D), B10S (10E) and B10M (10F).

FIG. 11 represents a linear restriction map for the pBS-SKT-S1.S2.Splasmid.

EXAMPLE 1

Induction of Antibodies Against a Hepatitis B Surface Antigen bySequential Injection of Bupivacaine and of a Plasmid Carrying a GeneCoding for the Antigen

1) Materials and Methods

1.1 Bupivacaine Pretreatment

All experiments were made on the muscles of the anterior tibia (AT) ofmice C57BL/6J aged between 5 to 7 weeks.

A single degeneration-regeneration cycle of the muscle fibres is inducedin the muscles of the anterior tibia of non-anaesthetized mice, byintramuscular injection of 50 μl marcaine (bupivacaine 0.5%, DMSO 1%)sold by Laboratoires Astra, France. The solution is injected using atuberculosis syringe with a needle fitted into a polyethylene sleeve, inorder to limit the penetration depth to 2 mm.

As marcaine is an anesthetic, injections into the right and left legswere performed at 10 to 30 minute intervals to prevent an overdose.

1.2 DNA Preparation

The plasmid used was constructed by cloning into a modified pBlueScriptvector of the Xho I-Bgl II restriction fragment of the pCP10 plasmidwhich contains the gene coding for the HBS surface antigen and thenon-translated sequences, both upstream and downstream, including thepolyadenylation signal.

The S gene was then recovered by digestion using KpnI-BssHII enzymes andthe fragment was cloned into the site of the pRC/CMV vector sold by InVitrogen. The final plasmid construction was called pCMV-HBS and wasfiled under N^(o) I-1370 with the CNCM.

This plasmid is represented schematically in FIG. 1. The CMV promoter issituated between the 288 nucleotide which is the cleavage position ofMluI and the 896 nucleotide, which is the cleavage position of KpnI. TheDNA fragment including the structural gene of the HBs antigen structurewas cloned between the 896 and 2852 nucleotides (position of BssH III).

The HBs gene spreads between the 911 (XhoI position) and 2768nucleotides (Bgl II position) respectively.

The complete sequence for this plasmid is sequence SEQ ID N^(o) 1.

The purified plasmid DNA was prepared by standard methods thenredissolved in PBS buffer and stored at −20° C. until the injection wasperformed.

1.3 DNA Injection

One to five days after the marcaine injection, DNA was injected into thesame area, the mouse being anaesthetized using sodium pentobarbital (75mg/kg interperitonal path).

The DNA solution which contains 50 μg of plasmid DNA and 50 μl of PBSbuffer was injected by a single intramuscular injection through the skininto the anterior tibia muscles undergoing regeneration.

The injections were performed bilaterally into the two legs of the mice,each animal thus receiving a total of 100 μg of recombinant plasmid DNA.As for the marcaine injection, the DNA solution was injected using thetuberculosis syringe with the needle described previously.

A single intramuscular DNA injection was performed in each leg.

2. Results

The results obtained are summarized in Table I below.

They show very clearly that a DNA injection after treatment withmarcaine allows a large number of seric antibodies to be obtainedagainst the hepatitis B surface antigen.

These results are surprising, from the analysis of the state of the artit was not inferred that a plasmid would allow the induction of anti-HBsantibodies which could be found in the serum and thus allow an effectivevaccination.

The ease of application of the plasmid vaccination, and the fact thatboosters would not be necessary, allows the consideration of a largescale vaccination.

EXAMPLE 2

Comparison of the Efficiency of a Plasmid Injection in the Presence andAbsence of Lipids.

A dose of 10 μg plasmid DNA from the SV40-luciferase vector availablecommercially (“pGL2-Control Vector” from Promega, reference E1 11) in 50μl of physiological solution was injected into the sucrose pretreatedmuscle following the method of David et al. (Hum. Gene Ther. 4:151-159(1993)). The injected DNA is mixed earlier with lipids such asdioctadecylamidoglycyl spermine (DOGS) or the following mixtures:DOGS+spermidine, and DOGS+polyethyleneglycol (PEG). The luciferaseactivity was determined 5 days after the injection.

These results are shown in table II below.

They show that the presence of lipids (DOGS) very reduces significantlythe efficiency of the plasmid injection with respect to a compositionwith no lipids (control).

EXAMPLE 3

Comparison of the Responses of Mice and Rabbits to Plasmids CarryingDifferent Promoters and Envelope Genes for the HBV Virus.

Four plasmids were constructed allowing the expression of one, two orthree envelope proteins for the HBV virus. In three of the constructions(pCMVBH-S, pCMVHB-S2.S, pCMVHB-S1.S2.S) the genes coding for the HBVvirus envelope proteins are put under transcriptional control of thepromoter of the CMV virus precursor genes (FIG. 1, FIG. 2A to 2C, FIGS.3 and 4). The fourth plasmid (pHBV-S1.S2.S) uses the promoter for theHBV virus surface genes contained in the pre-S1 region of this virus(Cattaneo et al. (1983) Nature, 305, 336) (FIG. 2D) as a transcriptionalcontrolling element. In the four constructions, the polyadenylationsignal used is contained in the HBV sequences present in 3′ of the Sgene.

1. In Vitro Control of the Vector Efficiency.

To control the efficiency of these vectors in vitro in eucaryote cells,mouse fibroblasts or myoblasts were transfected. A plasmid expressingthe three envelope proteins under control of the SV40 promoter (pSVS)was used as a control (Michel et al. 1984, Proc. Natl. Acad. Sci. USA,81, 7708-7712)). FIG. 6 illustrates the secretion kinetics of the HEsparticles in the culture supernatants. The low antigen levels producedby transfection of the pCMVHB-S1.S2.S vector are compatible with a largedegree of synthesis of the large envelope protein starting from the CMVpromoter. This protein being myristilised in its amino terminal region,is retained in the endoplasmic reticulum (Ganem, (1991), Current Topicsin Microbiology and Immunology, 168, 61-83). Retention in the cell ofproteins carrying the pre-S1 determinants was confirmed byimmunofluorescence.

The composition of the secreted particles was analyzed in an ELISAsandwich system using as capture antibodies a monoclonal mouse antibodyspecific to the pre-S1 (FIG. 7A) or pre-S2 (FIG. 7B) determinants ascapture antibodies and rabbit anti-HBs polyclonal serum as secondantibodies. These experiments show that the AgHBs particles producedstarting from pCMVHB-S1.S2.S vector carry pre-S1 and Pre-S2 determinantsshowing the presence of the large and medium envelope proteins of theHBV virus. Particles secreted after the transfection of the pCMVHB-S2.Sand pHBV-S1.S2.S vectors carry, in addition to the HBs determinants,pre-S2 determinants characteristic of the medium envelope proteins.

2) DNA Inoculation

DNA purified on a Quiagen column was injected by an intramuscular pathin a single injection of 100 μg (50 μg/leg) in the anterior tibia muscleof mice C57/BL6 (8 mice per group). Five days prior to the injection,the muscle was pretreated with cardiotoxin in order to inducedegeneration followed by regeneration of the muscles cells thus favoringthe DNA capture by these cells.

The DNA injection experiments were also carried out for rabbits. In thiscase, pCMVHB-S DNA was administered into normal muscle withoutdegeneration, either by using an injection gun without needle calledBiojector^(R), or by conventional syringes fitted with needles.

3) Anti-Hbs Responses for Mice Vaccinated with DNA

An anti-HBs antibody response is induced by a single injection of one orother of the four plasmids used.

The antibody response was analyzed using a commercial anti-HBsantibodies detection kit (Monolisa anti-HBs, Diagnostic Pasteur).Anti-preS2 antibodies are detected by an ELISA system using, on thesolid phase, a peptide from the pre-S2 (AA 120-145) region on the solidphase corresponding to a B major epitope carried by this area (Neuarthet al., (1985), Nature, 315, 154).

FIGS. 8A to 8D illustrate the anti-HBs (HBs-Ab) response kineticsexpressed in milli-international units/ml and the anti-pre-S2 response(pres2Ab) determined as optical density (492 nm) for 1/100 dilutedserums. Detection was carried out using a mouse anti-immunoglobulinantibody (IgG) coded with peroxidase.

The injection of the pCMVHB-S plasmid (FIG. 8A) induces a constantanti-HBs antibody synthesis. Seroconversion was observed in 100% of micefrom one week after the injection with an antibody average level of 48mUI/ml (from 12 to 84 mUI/ml, standard deviation (SD)=28), which is 4 to5 times superior to the threshold required in man to provide protection(10 mUI/ml).

The induced response for a single injection of pCMVHB-S2.S plasmid (FIG.8B) is characterized by the very early apparition of anti-HBsantibodies. These antibodies reach an average level of 439 mUI/ml (from104 to 835 mUI/ml; SD=227) at one week then reduce before increasingagain to reach the initial level at 13 weeks. The significance of thisantibody peak will be discussed later. A peak for anti-pre-S2 IgGantibodies is observed at two weeks.

The appearance of anti-HBs antibodies induced by injection ofpCMVHBV-S1.S2.S plasmids. (FIG. 8C) and pHBV-S1.S2.S (FIG. 8D) isslightly delayed as the mice only seroconvert to 100% after two weeks.The seroconversion profile is identical, it is characterized by aninitial response which is specific to the pre-S2 antigen followed by ananti-HBs response which gradually increases to reach a level of 488mUI/ml (from 91 to 1034 mUI/ml; SD=552) (pCMVHB-S1.S2.S) and 1725 mUI/ml(from 143 to 6037 mUI/ml; SD=1808) (pHBV-S1.S2.S) at 13 weeks.

4) Anti-HBS Response of Rabbits Injected with DNA

Results presented in tables III and IV show that the antibody levelsdetected at 8 weeks in rabbits immunized using the Biojector aresignificantly higher than those obtained by a DNA injection by needle.

5) Qualitative Analysis of the Humoral Response

ELISA systems applied to the solid phase of the HBs antigens of varyingcomposition with respect to the determinants presented on the solidphase and using mouse antibodies specific to IgM or IgG as secondantibodies gave a qualitative analysis of the antibody response that wasachieved.

In all cases, the single injection of DNA in mice is characterized bythe early appearance of AgHBs specific IgM followed immediately byconversion to IgG isotype antibodies which is characteristic of thememory response induced by the auxiliary T cells. The antibody responseto the DNA injection is characterized by its prematurity. Indeed,seroconversion is achieved 8 to 15 days after the injection depending onthe DNA type used and in all cases the plateau is achieved in four weeksand maintained constantly over a period of 12 weeks.

The use of the heterologous sub-type HBs antigens (ad) fixed on ELISAplates allows the formation/detection of the presence, in the serum ofimmunized mice, of antibodies specific to the anti-a group, and bydifference in reactivity with respect to AgHBs of the same sub-type(ay), of antibodies specific to the anti-y sub-type. The presence ofantibodies specific to determinants of the AgHBs group is very importantas the former are capable of giving protection against the heterologoussub-type virus during virulent tests in chimpanzees (Szmuness et al.(1982) N. Engl. J. Med. 307, 1481-1486)

Analysis of the response induced by the pCMV-S2.S vector shows that ithas a remarkable similarity with the one which can be observed in manduring infection. It is characterized by an extremely early (8 days)peak for IgM which is specific to the pre-S2 region immediately followedby conversion to anti-pre-S2 IgG (FIG. 9). This response is followed bythe appearance of IgM then IgG anti-HBs antibodies. The anti-HBsantibody production is constant and reaches a maximum after 4 weeks. At13 weeks IgG anti-HBS and anti-pre-S2 remain at a constant level.

The anti-sub-group (y) response precedes that of the anti-group response(a) in the same way as that described for the vaccination with therecombinant vaccine (Tron et al., (J. Infect. Dis. 160, 199-204).

The response obtained with the three other DNA vaccines illustrates thecommutation of class IgM→IgG which is characteristic of the secondaryresponse. The response being first of all directed against the sub-typebefore being against the AgHBs group determinants.

The long term response which was studied for pCMVHB-S DNA shows that theantibody peak is reached within 3 months and this remains at a constantlevel 6 months later (Table V).

6. Genetic Vaccine and Non-response

The high number of non-responders to the classical vaccine (2.5 to 5)remains a major problem for vaccination against hepatitis B. It has beenpossible to correlate the non-response in man to certain HLA types(Krustall et al., (1992) J. Exp. Med. 175, 495-502) and to a defect inthe antigen presentation or stimulation of the auxiliary T cells.

To study the possible impact of the genetic vaccination on the AgHBsnon-response, a range of mice strains were used for which the responseto various HBV virus envelope proteins is controlled genetically and hasbeen well characterized by Millich et al. (1986 J. Immunol. 137, 315).The pCMVHB-S construction previously described was injected into B10(H-2^(b)) B10.S (H-2^(s)) and B10.M (H-2^(f)) mice muscles.

The B10 strain responds to the three virus envelope proteins, the B10.Sstrain does not respond to AgHBs but this non-response can be overcomeby immunization with HBsAg antigens which are carrying pre-S2determinants. The B10M strain is totally non-responsive to both HBs andpre-S2 antigens. A response for the latter strain can be achieved byimmunization using AgHBs carrying pre-S1 determinants.

The mice immunized by the DNA received a single injection (100 μg) inthe regenerating muscle. Control mice were injected with twointraperitonal injections of protein at an interval of one month, thefirst of 2 μg AgHBs to which the complete Freund additive (CFA) wasadded and the second of 2 μg AgHBs to which the incomplete Freundadditive (IFA) was added.

The results obtained for pCMVHB-S are illustrated by FIGS. 10A to 10F.

-   -   In the B10 strain (good responder) the DNA induced response is        earlier than that induced by the protein after a single        injection.    -   The appearance of anti-HBs antibodies sub-type specific then        group specific after immunization with pCMVHB-S DNA was observed        in the B10S strain (non-responder to AgHBS in the absence of        pre-S2). Group specific anti-HBs antibodies are observed in HBs        protein immunized mice only after the second injection.    -   A group and sub-type specific anti-HBs response is obtained for        DNA immunization of strain B10M (non-responder to AgHBs in        absence of pre-S1) whereas only a sub-type specific response is        induced by the protein with two injections being required.

The response induced by the three vector types is compared in the threemice strains.

CONCLUSIONS

It is generally thought that the humoral response to HBs antigens issufficient by itself to give protection. The presence of antibodiesdirected against other determinants (pre-S1 and pre-S2) carried by thevirus envelope proteins, themselves protectors, could improve theresponse quality. The experiments reported here as a whole illustratesthat the humoral response induced by the genetic anti-hepatitis Bvaccination is greater in several fields than that which can be achievedfor the classical vaccination.

In terms of seroconversion levels: the 100% level is obtained, afteronly one injection, from day 8 for mice immunized with pCMV-HBS DNA andpCMVHB-S2.S.

In terms of response level: the 10 mUI/ml threshold level, consideredsufficient to give protection in man, is always greatly exceeded.

In terms of the speed of response: in 8 days a very high level ofanti-pre-S2 antibodies is obtained for the pCMVHB-S2.S vector and it isknown that the former are capable of giving protection by themselves(Itoh et al., (1986) Proc. Natl. Acad. Sci. USA 83, 9174-9178).

In terms of response stability: anti-HBs anti-bodies remain constant ata high level for more than 6 months.

In terms of response quality: type IgG antibodies characteristic of aresponse which is dependent on the auxiliary T cells and therefore on amemory response are obtained.

In terms of anti-viral activity: the antibodies are specific to theviral sub-type but especially group specific and therefore susceptibleto giving a cross protection.

In terms of biological significance: the response profile obtained bypCMVHB-S2.S immunization mimes totally that which is observed in manafter a resolved viral infection.

Deposits: Nucleotide plasmid vectors pCMV/HBS, pRSV/HBS, and pCMV/HB-S1.S2.S, pHBV-S1.S2.S, and pCMVHB-S2.S were deposited on Oct. 21, 1993with the Collection Nationale des Cultures des Micro-organismes de1′Institut Pasteur (CNCM) under accession numbers I-1370, I-1371,I-1411,I-1409, and I-1410, respectively. Applicants' assignee, theUniversity of Ottawa, represents that the CNCM is a depository affordingpermanence of the deposit and ready accessibility thereto by the publicif a patent is granted. All restrictions on the availability to thepublic of the material so deposited will be irrevocably removed upon thegranting of a patent. The material will be available during the pendencyof the patent application to one determined by the Commissioner to beentitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The depositedmaterial will be maintained with all the care necessary to keep itviable and uncontaminated for a period of at least five years after themost recent request for the furnishing of a sample of the depositedvector, and in any case, for a period of at least thirty (30) yearsafter the date of deposit or for the enforceable life of the patent,whichever period is longer. Applicants acknowledge their duty to replacethe deposit should the depository be unable to furnish a sample whenrequested due to the condition of the deposit.

TABLE I Induction of antibodies against the hepatitis B surface antigenLevel of antibodies against hepatitis B surface antigen in the serum(mIU/ml) Number Before DNA 15 days after 35 days after Description ofmice injection DNA injection DNA injection DNA 5 0 average: 56 fromaverage: 59 injected  5 to > 140 1 day after marcaine treatment DNA 5 0average: 71 from average: 47 injected 21 to > 108 5 days after marcainetreatment

TABLE II Luciferase RLU/sec/muscle (Average ± Percentage SEM) RLU =Relative relative to the Group Light Unit control Control 43 082 ± 5 419 100% 4X DOGS 28 ± 7 0.06% DOGS - Spermidine  50 ± 23 0.12% PEG-DOGS  0± 0 0.00%

TABLE III Immunization with the Biojector^(R) pCMV-HB.S N^(o) 0 weeks 2weeks 8 weeks 2.1 0 517 380 2.2 0 374 322 3.1 0 258 418 4.1 0 400 40454.2 0 88 86 4.3 0 314 420 6.1 0 415 1001 6.2 0 1543 3517 6.3 0 1181 141Average 0 566 mUI/ml 1148 mUI/ml SD 0 476 1521 SEM 0 159 507 N 9 9 9 CV84% 133%

TABLE IV Immunization by injection using a needle pCMV-HB.S N^(o) 0weeks 2 weeks 8 weeks 1.1 1 0 1 5.1 0 287 186 5.2 0 162 798 5.3 0 305203 7.1 0 86 175 7.2 0 1108 dead Average 0 325 mUI/ml 273 mUI/ml SD 0401 305 SEM 0 164 136 N 6 6 5 CV 245% 124% 112%

TABLE V Long term response of a mouse vaccinated with pCMVHB-S 1 month 2months 3 months 6 months * a-HBs titre 227 662 1299 1082 in mUI/ml *a-HBs ELISA 3.5 × 10⁻⁴ 5 × 10⁻⁴ 8.5 × 10⁻⁴ 9 × 10⁻⁴ titre

1. A method of inducing an antigen specific immune response in amammalian subject comprising administering to the mammalian subject anexpression plasmid vector that comprises a gene encoding a hepatitis Bvirus surface or core antigen, or a fragment thereof, and a promoter forexpression of the gene in the subject, wherein the gene is expressed inthe mammalian subject in an effective amount to induce an antigenspecific immune response against the hepatitis B virus surface or coreantigen.
 2. The method of claim 1, wherein administration of said vectoris conducted at least five days after administration of at least onesubstance capable of inducing a coagulating necrosis of muscle fibersand wherein said administration of said vector and said substance isabout in the same area.
 3. The method of claim 2, wherein said substanceis bupivacaine.
 4. The method of claim 3, wherein the vector isadministered at least 7 days after the administration of bupivacaine. 5.The method of claim 1, wherein the administration is carried out byintramuscular injection.
 6. The method according to claim 5, wherein theintramuscular injection is carried out using a liquid jet gun.
 7. Themethod of claim 1, wherein the promoter is endogenous to hepatitis Bvirus.
 8. The method of claim 1, wherein the antigen is a protein orantigenic portion thereof selected from the group consisting ofmajor/small envelope protein (S), middle envelope protein (S₂₋-S), andlarge envelope protein (S₁-S₂₋-S).
 9. The method of claim 8, wherein thegene encodes the S protein.
 10. The method of claim 1, wherein thepromoter is a viral promoter.
 11. The method of claim 10, wherein thepromoter is a cytomegalovirus promoter.
 12. The method of claim 1,wherein the promoter is a mammalian promoter.
 13. The method of claim 1,wherein the vector is pCMV-HB-S1.S.S deposited with the CNCM under No.I-1411.
 14. The method of claim 1, wherein the vector is pCMV-HB-S2.Sdeposited with the CNCM under No. I-1410.
 15. The method of claim 1,wherein the vector is pRSV-HBS deposited with the CNCM under No. I-1371.16. The method of claim 1, wherein the vector is pHBV-S1.S2.S depositedwith the CNCM under No. I-1409.